The adoptive transfer of T cell receptor (TCR) engineered T cells and hematopoietic stem cells (HSCs) can potentially provide a patient with a large supply of tumor-specific T cells, enhancing immune-based therapies for cancer. A full understanding of the basic biology of these TCR engineered cells will be very valuable to help design a more potent therapy. In this project, we propose to use the B16 melanoma tumor model to conduct a comprehensive study of the Pmel TCR engineered peripheral CDS T cells, CD4 T cells and HSCs. For this purpose, retroviral and lentiviral vectors will be constructed to co-deliver the genes encoding the Pmel TCR and a bioluminescence reporter into the target cells. The in vivo fate of the TCR-engineered T cells and HSCs will be monitored using Bioluminescence Imaging (BLI) in a live animal in real-time. We plan to study the antimelanoma immunity generated by the adoptive transfer of TCR-engineered T cells (Specific Aim1), HSCs (Specific Aim2), and their combination (Specific Aim3). Various conditions for the in vitro TCR transduction, recipient animal pre-conditioning, adoptive transfer, and post-transfer immunization will be evaluated for their contribution to an effective therapy. In particular, we will study the synergy between the anti-tumor CD4 and CDS T cells, and the influence of the CD4+CD25+ regulatory T cells (Tregs) for adoptive therapy. We will also try to enhance the anti-tumor effectiveness of the engineered anti-tumor T cells by co-delivery of an enhancement gene together with the TCR genes into the target cells. From the proposed study, we expect to gain a thorough understanding of the basic biology of TCR-engineered peripheral T cells and HSCs, and the anti-tumor immunity generated through adoptive transfer of these engineered cells. This basic biology will inform the clinical investigations being conducted in Project 1 and Project 3.